Cytokine-Sensitive Replication of Hepatitis B Virus in Immortalized Mouse Hepatocyte Cultures

Abstract

We have previously shown that alpha/beta interferon (IFN-␣/␤) and gamma interferon (IFN-␥) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-␥ and tumor necrosis factor alpha (TNF-␣) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-␣/␤, IFN-␥, and TNF-␣ at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-␣/␤ or IFN-␥, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-␥, but not IFN-␣/␤, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-␣. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.Hepatitis B virus (HBV) is a hepatotropic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (5). We have previously shown that the intrahepatic induction of alpha/beta interferon (IFN-␣/␤), gamma interferon (IFN-␥), and tumor necrosis factor alpha (TNF-␣) by various stimuli can inhibit HBV gene expression and/or HBV replication in the livers of HBV-transgenic mice (3,9,10,23). More recently, we demonstrated that similar noncytopathic antiviral events probably occur in the livers of chimpanzees acutely infected with HBV (15).Although it is now well documented that HBV replication can be inhibited by these cytokines (11,19), in vivo systems are by nature difficult to manipulate and control. Therefore, studies designed to determine which cytokines primarily inhibit HBV replication and to decipher the antiviral intracellular mechanism(s) they induce would be greatly facilitated by an in vitro cell culture system that accurately reflects the cytokinedependent control of HBV observed in vivo. Unfortunately, primary cultures of HBV-transgenic hepatocytes are not suitable for these studies because they rapidly become less well differentiated in vitro. Recently, however, well-differentiated immortalized mouse hepatocyte cell lines were established from transgenic mice expressing the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met) in their livers (2). Cells prepared from these mice are not transformed, and they can be induced to a stable, highly differentiated hepatocyte phenotype by dimethyl sulfoxide (DMSO) (29).In this report, we describe the establishment and characterization of an i...

Tumor Eradication by Hepatitis B Virus X Antigen-Specific CD8+ T Cells in Xenografted Nude Mice

“…We speculate that this result may arise in two ways: 1) tumor eradication may result from direct killing mediated by Ag-specific CD8 ϩ T cells, and 2) IFN-␣/␤-, IFN-␥-, or TNF-␣-mediated killing may occur because the cells are able to secrete these cytokines. Similar observations have been reported (37)(38)(39).…”

PD-1:PD-L1 Interactions Contribute to the Functional Suppression of Virus-Specific CD8+ T Lymphocytes in the Liver

“…Cells were maintained in hepatocyte culture medium (RPMI 1640 (Mediatech), 10% FCS (Invitrogen Life Technologies), 55 ng/ml epidermal growth factor (Calbiochem), 16 ng/ml insulin growth factor-II (Calbiochem), 10 g/ml insulin (SigmaAldrich), and penicillin-streptomycin-glutamine (100ϫ; liquid) (Invitrogen Life Technologies)), as previously described (30). For treatment with CTLconditioned medium, cells were plated 24 h before treatment in collagencoated 60-mm dishes.…”

Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs

“…23. Cells were plated on collagen I-coated dishes (Becton Dickinson) in RPMI medium 1640 supplemented with 10% FCS (Invitrogen), 55 ng͞ml EGF (Becton Dickinson), 16 ng͞ml insulin-like growth factor II (Calbiochem), 10 g͞ml insulin (Sigma), and penicillinstreptomycin-glutamine (Invitrogen), as described in ref.…”

“…To test the effectiveness of the resulting adenovirus-expressed shRNAs, we infected duplicate cultures of a highly differentiated HBV-positive hepatocyte cell line derived from the 1.3.46 HBV transgenic mice (23). Total RNA was extracted from cells 72 h postinfection to determine the level of HBV RNA.…”